WO1989012095A1 - Procede d'evaluation de vaccins recombinants contre le virus de l'immunodeficience - Google Patents

Procede d'evaluation de vaccins recombinants contre le virus de l'immunodeficience Download PDF

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WO1989012095A1
WO1989012095A1 PCT/US1989/002485 US8902485W WO8912095A1 WO 1989012095 A1 WO1989012095 A1 WO 1989012095A1 US 8902485 W US8902485 W US 8902485W WO 8912095 A1 WO8912095 A1 WO 8912095A1
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siv
virus
vaccinia
promoter
gene
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PCT/US1989/002485
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Linda R. Gritz
Virginia Stallard
Dennis L. Panicall
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Applied Biotechnology, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HIV Human Immunodeficiency Virus
  • HIV exhibits a remarkable ability to persist and eventually induce chronic disease in spite of host immune defenses.
  • an effective vaccine against AIDS may have to prevent totally the establishment of any foci of infected cells in the human host. This goal is made especially difficult by the great genetic variability of HIV, particularly in the gene that codes for the surface glycoproteins present in the outer membrane of the virus.
  • an effective vaccine will need to stimulate both humoral and cell-mediated immunity.
  • CTLs Cytotoxic T-Lymphocytes
  • CTLs can recognize "conserved” regions of viral proteins expressed on the surface of infected cells, including non-envelope viral proteins that are not displayed on the surface of the mature virion particles.
  • these other viral proteins show a constancy in their structure and associated antigens.
  • CTL-mediated functions may be necessary to obtain broad-spectrum resistance to the many HIV variants.
  • CTL and NK natural killer cells
  • NK natural killer cells
  • the ability of both CTL and NK (natural killer cells) to eliminate virus-infected cells may curtail the spread of infection by preventing cell-to-cell transmission and the resulting propagation of HIV infection. It is important, therefore, to optimize the probability of eliciting protective immunity by developing vaccines with the potential of stimulating both antibody and cell-mediated responses.
  • Conventional approaches to vaccine development rely upon the use of live, attenuated or killed forms of the virus as immunogens. The success of these approaches in the past was due in part to the presentation of native antigens and the ability of attenuated virus to elicit the complete range of immune responses obtained in natural infection. There are some obvious difficulties with the use of whole virus for an HIV vaccine.
  • peptide vaccines consisting of chemically synthesized, immunoreactive epitopes
  • subunit vaccines produced by expression of viral proteins in recombinant heterologous cells
  • live viral vectors for the presentation of one or a number of defined antigens.
  • peptide and subunit vaccines are subject to a number of potential limitations. A major problem is the difficulty of ensuring that the conformation of the engineered proteins mimics that of the antigens in their natural environment.
  • Suitable adjuvants and, in the case of peptides, carrier proteins, must be used to boost the immune response.
  • peptide and subunit vaccines elicit primarily humoral responses and thus may fail to evoke effective cell-mediated immunity.
  • the gene product has been shown to be functional by virtue of its ability to induce CD4-dependent cell fusion leading to cell death, one of the characteristic manifestations of HIV cytopathology (Lifson et al. (1986) Nature 323:725-728; Papovic et al.
  • Chimpanzees are the only nonhuman primate infectable with HIV and thus constitute the only vaccine-challenge system that has been utilized to date.
  • chimpanzees become infected following HIV inoculations, they do not develop any AIDS-like illness. Consequently, their response to a vaccine many not be predictive of the vaccine's effect in humans.
  • their very limited numbers will severely restrict their further use for such studies. Testing of numerous vaccines and the variables associated with each will simply not be possible in chimpanzees.
  • SIV sem immunodeficiency virus
  • This invention pertains to recombinant pox viruses capable of expressing simian immunodeficiency virus antigens, particularly antigens capable of eliciting a protective immune response against simian immunodeficiency virus (SIV) and to methods of producing the recombinant pox virus.
  • This invention also pertains to intermediate DNA vectors which recombine with pox virus in vivo to produce the modified pox viruses, to methods of vaccinating a non-human primate host with the recombinant pox virus to elicit protective immunity against SIV in the host, and to use of non-human primates as a model system to evaluate the effectiveness of pox virus vaccines containing SIV antigens.
  • this invention pertains to the application of the information obtained using the SIV/ non-human primate model system in the development of pox virus-based vaccines for the prevention of human immunodeficiency virus (HIV) infection of humans.
  • HIV human immunodeficiency virus
  • Recombinant pox viruses capable of expressing SIV antigens are produced by integrating into the pox virus genome a gene or genes encoding the SIV antigen (s) of interest.
  • the SIV gene(s) is inserted into a region of the pox virus genome which is nonessential for replication of the pox virus.
  • Each gene is inserted into the pox virus genome in association with a pox virus promoter to direct its expression.
  • the SIV gene(s) is integrated into the pox viral genome by an in vivo recombination event between the pox virus and an intermediate DNA vector carrying the SIV gene.
  • the intermediate DNA vector contains the SIV gene linked to a pox viral promoter located within a DNA sequence homologous to a region of the pox viral genome which is nonessential for replication of the pox virus.
  • the intermediate vector must also have the capacity to replicate in a prokaryotic host; amplification of the vector in a prokaryotic host cell allows for the production of necessary amounts of vector DNA for in vivo recombination.
  • the vector in order to detect the presence of the intermediate plasmid vector in the prokaryotic host, the vector must also contain a gene which confers a selectable phenotypic trait on the prokaryotic host cell.
  • the vector comprises: a. a prokaryotic origin of replication; b. a gene encoding a marker for selection in the prokaryotic host; c. a pox viral promoter; d. a gene encoding a simian immunodeficiency virus antigen under the direction of the pox viral promoter; e. DNA sequences of the pox virus into which the gene encoding the SIV antigen is to be integrated. These DNA sequences flank the promoter and structural gene at both the 5' and 3' ends and are homologous to the region of the pox virus genome where the SIV gene is to be inserted.
  • Recombination of the DNA vector and the pox virus is achieved in an appropriate host cell.
  • Appropriate host cells for in vivo recombination are eukaryotic cells which are 1) transfectable by the DNA vector and 2) infectable by pox virus. The host cell is infected with the pox virus and then is transfected with the DNA vector. Virus is allowed to replicate in the host cell and recombination occurs in vivo, resulting in insertion of the SIV gene into the pox virus genome.
  • the viral progeny is isolated away from the wild type virus.
  • expression of the selectable marker provides a basis for selection of recombinant virus containing integrated SIV DNA.
  • Other methods of selection include detection of integrated SIV gene by hybridization with homologous DNA probes or selection for absence of the product of the viral gene into which the DNA vector has been inserted.
  • the recombinant virus is a virus which expresses in tissue culture and in an inoculated animal host the SIV antigen(s) of interest.
  • the antigen expressed by the recombinant pox virus will elicit cell-mediated and humoral responses which may be capable of recognizing and neutralizing the SIV from which the antigen is derived.
  • certain antigens which are shared antigenic determinants may provide cross-protection against the various simian viruses.
  • the DNA vector for recombination with pox virus also contains a gene encoding a selectable marker which permits selection of viral recombinants containing the inserted SIV DNA.
  • the vector will contain these additional elements located between the flanking pox viral sequences: f. a second pox viral promoter; and g. a gene encoding a selectable marker, the gene being under the direction of the second pox viral promoter.
  • This invention also pertains to methods of using a simian model to evaluate the effectiveness with which recombinant pox viruses expressing SIV antigens protect monkeys from acquiring an SIV infection and developing Simian AIDS, in order to identify HIV homologs effective in treating or preventing AIDS in humans.
  • the preferable monkey to be used in this model system is the macaque monkey.
  • SIV pox-viral recombinants . are tested for their ability to protect macaques from SIV infection.
  • the extremely close correspondence between SIV and HIV genomic material makes it likely that information obtained from work on the simian model system will be applicable to the design of a safe and effective pox virus-based HIV vaccines for human use.
  • this invention also pertains to the development of DNA vectors and pox virus containing HIV counterparts of SIV DNA sequences.
  • Vaccination with a live virus will stimulate cell-mediated and humoral immunity.
  • the proteins that are expressed are expected to be appropriately modified, and if the required signals are present, they may also be localized to the proper regions of the cell or cellular membrane (Stephans et al. (1986) EMBO J. 5:237).
  • vaccinia virus has been shown to be stable upon storage under normal field conditions. The vaccine can also be administered relatively simply.
  • a recombinant vaccine utilizing vaccinia virus would avoid the problems associated with vaccinating with live attenuated or killed virus, namely the possibility that vaccines vased on whole viral pathogens may not be properly killed or may revert to virulence. It is expected that live viral vaccines would also be more immunogenic than subunit vaccines.
  • Figure 1 shows the similar genomic organization of Simian Immunodeficiency Virus (SIV) and Human Immunodeficiency Virus (HIV-1).
  • Figure 2 shows the construction of pAbT4500 and pAbT4525, plasmid vectors for insertion of foreign genes into vaccinia by in vivo recombination (IVR) containing the 30k or 40k promoter, respectively, the vaccinia thymidine kinase gene region for directing the recombination, a lacZ gene under the control of the vaccinia BamF promoter, and a bacterial replicon and ampicillin resistance gene.
  • IVR in vivo recombination
  • Figure 3 shows the construction of pAbT4537, identical to pAbT4525 except that the thymidine kinase region has been shortened and modified to contain additional, convenient restriction sites.
  • Figure 4 shows the construction of IVR vector pAbT4554, which contains the vaccinia HindlllM region for directing recombination, the 29k host range gene for selection of recombinants, the vaccinia 30k promoter and multiple cloning site for insertion of foreign genes, and a bacterial replicon and ampicillin resistance gene.
  • Figure 5 shows the construction of IVR vectors pAbT4555 and pAbT4556, which are identical to pAbT4554, except that pAbT4555 contains two promoters, 30k and 40k, and pAbT4556 contains three promoters, 30k, 40k and 7.5k, each followed by a multiple cloning site for the insertion of foreign genes.
  • Figure 6 shows the construction of IVR vector pAbT4586, which is identical to pAbt4537, except that pAbT4586 contains two vaccinia promoters, 7.5k and 40k, each followed by a multiple cloning site for the insertion of foreign genes.
  • Figure 7 shows the construction of plasmid pAbT4572, a plasmid vector for the insertion of the SIVMAC-251 env gene into vaccinia, with the env gene under the control of the vaccinia 40k promoter.
  • Figure 8 shows the construction of plasmids pAbT4575 and pAbT 4577.
  • pAbT4575 is an IVR vector for the insertion of the SIV MAC-251 gag-prot gene into vaccinia with the gag-prot gene under the control of the 40k promoter.
  • pAbT4577 is an IVR vector for the insertion of the SIV MAC-251 env and gag-prot genes into vaccinia, with the env gene under the control of the 30k promoter and the gag-prot gene under the control of the 40k promoter.
  • Figure 9 shows the construction of plasmid pAbT4576, an IVR vector for the insertion of the
  • Figure 10 shows the construction of plasmid pAbT4581, an IVR vector for the insertion of the SIV MAC-251 sor gene into vaccinia, with the sor gene under the control of the 30k promoter.
  • Figure 11 shows the construction of plasmid pAbT4583, an IVR vector for the insertion of the
  • Figure 12 shows the construction of plasmid pAbT4585, an IVR vector for the insertion of the SIV MAC-251 env and gag-prot genes into vaccinia,. with the env gene under the control of the 40k promoter and the gag-prot gene under the control of the 7.5k promoter.
  • Figure 13 shows the construction of plasmid pAbT4589, an IVR vector for the insertion of the SIV MAC-251 env and gag-prot genes into vaccinia.
  • pAbT4589 is identical to pAbT4585, except that pAbT4589 contains elements for insertion at the vaccinia thymidine kinase region, whereas pAbT4585 contains elements for insertion and selection at the vaccinia HindlllM region.
  • Genes encoding simian immunodeficiency viral antigens can be obtained from the genomic cDNA of a simian immunodeficiency virus or from available subgenomic clones containing the genes.
  • the genomic organization of both the HIV and SIV genomes is represented diagrammatically in Figure 1.
  • the gene organization of the two viruses is remarkably similar. Homologs of each of the HIV-1 open reading frames are present in SIV. Because the structure of SIV is extremely similar to that of HIV, the assumption has been made that data from HIV can be extrapolated to SIV.
  • the HIV virion is composed of a core surrounded by a lipid membrane containing the envelope (env) glycoprotein. This bipartite envelope protein has been considered the most promising candidate on which to base a vaccine strategy.
  • the envelope glycoprotein is believed to play a key role in HlV-induced disease. It specifically reacts with the CD4+ surface antigen to mediate both virus-tocell and cell-to-cell transmission and, in addition, mediates a fusion reaction between infected cells which express the glycoprotein on the surface and CD4+ cells. This reaction, which is postulated to result in the formation of inviable, multinucleated syncytia as well as in the death of individual cells, may account in part for the progressive nature of HIV and possibly SIV disease.
  • the core particle of HIV which contains the viral RNA, as well as reverse transcriptase and integrase proteins, is surrounded by a protein shell or capsid.
  • the capsid proteins which are more highly conserved among strains than are the envelope proteins, are derived from the viral gag gene. These proteins are primarily located beneath the lipid envelope. The presence in infected patients of antibodies to the gag proteins of HIV may correlate with the stage of disease, with more seriously ill individuals exhibiting little or no anti-gag reactivity.
  • the gag proteins could serve as an important immunogens in a recombinant vaccine.
  • genes encoded by the HIV virus may also encode important immunogens appropriate for inclusion in a recombinant vaccine.
  • the pol gene like gag is well conserved among different isolates and encodes a polypeptide which is proteolytically cleaved into a protease, a reverse transcriptase, and an endonuclease.
  • Antibodies to the HIV-1 endonuclease and reverse transcriptase have been detected in infected individuals.
  • CTL specific for HIV-1 reverse transcriptase have been detected in HIV-1 infected individuals (Walker et al. (1988) Science 240:64-66).
  • the HIV-1 sor gene product is required for cell-free viral infection of cells. HIV strains in which the sor gene has been deleted are defective in their ability to transmit in a cell-free manner. Antibodies to the sor gene product have been detected in infected individuals; these antibodies could play a role in preventing infection of cells by HIV and, possibly, SIV viral products.
  • HIV and SIV also contain at least four additional genes.
  • the tat gene encodes a protein that functions as a potent trans-activator of HIV gene expression.
  • the art gene product also up-regulates HIV synthesis by a transacting antirepression mechanism.
  • the 3'-orf gene product may down-regulate virus expression.
  • the R gene codes for an immunogenic protein whose function is currently unknown.
  • SIV and HIV genes selected for expression in recombinant pox viruses of this invention, and their protein products, are outlined in Table 1.
  • gag and sor genes can be expressed in pox virus by insertion of the relevant coding region into the viruses under the direction of pox viral promoters.
  • the expression of gag and pol products is more complicated.
  • the gag precursor polypeptide p55
  • a gag-pol fusion polypeptide encoded in the -1 reading frame with reference to gag, which is processed to yield a protease, reverse transcriptase, and endonuclease.
  • Synthesis of the gag-pol fusion polypeptide occurs as a result of ribosomal framshifting; typically, retroviral gag and gag-pol precursor polypeptides are synthesized at a ratio of 10-20 to 1.
  • a recombinant pox virus can be created that contains the entire SIV or HIV gag-pol domain under the control of a single pox virus promoter.
  • the pattern of gag and pol expression in this recombinant will likely depend upon the efficiency of ribsomal frameshifting; if it is highly inefficient, the predominant protein product will be unprocessed gag precursor polypeptide.
  • a pox viral recombinant can be constructed in which separate promoters are used to direct the expression of the SIV gag and pol genes.
  • site directed mutagenesis can be used to modify the 5' end of the pol gene in order to permit its individual expression.
  • Recombinant pox viruses that express either SIV gag or pol are constructed.
  • a single recombinant that expresses both of these genes under the control of separate promoters can be constructed. 2 .
  • a suitable pox virus for generating recombinant viruses includes any member of the pox family which does not cause significant disease in normal humans or animals.
  • the preferred pox virus is vaccinia virus, a relatively benign virus which has been used for years as a vaccine against smallpox.
  • Vaccinia virus is capable of infecting non-human primates such as monkeys and thus can be evaluated in the model system of this invention.
  • General techniques for integration of heterologous DNA into vaccinia virus to provide modified vaccinia virus capable of expressing foreign protein encoded by the heterologous integrated DNA are described by Paoletti et al., U.S. Patent No. 4,603,112, the teachings of which are incorporated by reference herein.
  • This vector is capable of carrying either SIV genes or the HIV counterparts of the SIV genes (e.g., gag, pol, env, sor, 3' orf, art, tat).
  • SIV genes e.g., gag, pol, env, sor, 3' orf, art, tat.
  • foreign DNA sequences to be inserted into the vector are those of the SIV genome. It is understood that corresponding DNA sequences of the HIV genome will also be suitable.
  • the vector contains (i) a prokaryotic origin of replication so that the vector may be amplified in a prokaryotic host, (ii) a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector (e.g., a gene encoding antibiotic resistance) (iii) DNA sequences homologous to the region of the pox virus genome where SIV gene is to be inserted, (iv) one or more SIV genes inserted at a site within this pox virus sequence and (v) one or more pox virus regulatory sequences
  • the vector backbone can be derived from any of several plasmid vectors capable of replication in a prokaryotic host, e.g., pBR322 (Sutcliffe, Cold Spring Harbor Symp., Quant.
  • the vector preferably contains a gene which encodes a marker which will allow selection of recombinant pox viruses containing integrated SIV DNA.
  • the gene encoding the marker is placed under control of a pox virus promoter.
  • a preferred marker gene is the E.
  • beta-galactosidase coli lacZ gene which encodes the enzyme beta-galactosidase.
  • Recombinant pox virus will express beta-galactosidase along with the SIV antigen.
  • beta-galactosidase production is detected as an indication of insertion and expression of the foreign DNA.
  • a chromogenic indicator (BluoGal) recombinant viruses which express beta-galactosidase will form blue plaques while wild-type virus, which do not express beta-galactosidase will form clear plaques.
  • a recombinant virus that has sequences in- serted into the pox virus TK (thymidine kinase) gene will have an inactivated TK gene and will grow in the presence of BUdR, while virus containing a wild-type, uninterrupted TK gene will be unable to grow in the presence of BUdR.
  • TK thymidine kinase
  • Other selectable markers include genes which confer antibiotic resistance in an infected host cell, e.g., the Neo R gene. Infected cells are grown in media containing the antibiotic at a concentration toxic for antibiotic sensitive cells. Under these conditions, cells infected with a recombinant virus expressing the resistance marker will produce virus. Cells infected with virus not containing the marker will not produce virus.
  • Another selection procedure relies upon a single vaccinia-encoded function. This procedure obviates the need for the use of drugs, mutagens, or chromogenic indicators, and permits rapid (2-3 wk) purification of recombinant viruses.
  • the method is described in co-pending U.S. patent application Serial No. 205,189 , entitled “Method Of Selecting For Recombinant Pox Viruses", Attorney's docket number ABT88-02, filed concurrently with this application, the teachings of which are incorporated herein by reference, Briefly, a vaccinia virus is employed that contains a mutation in a specific structural gene (29k gene), located in the HindlllM fragment of vaccinia virus.
  • the intermediate DNA vector contains vaccinia DNA sequences capable of restoring the mutant gene function; these sequences also direct recombination to the site of the mutant gene in the HindlllM region.
  • recombinant vaccinia viruses regain the ability to grow on RK13 cells, and can be isolated from non-recombinant viruses, which are unable to grow on these cells.
  • a preferred DNA vector for recombination with the preferred vaccinia virus comprises: a.
  • vaccinia promoters e.g., the vaccinia UK, 7.5K, 30K, 40K or BamF promoter or modified versions of these promoters
  • structural genes encoding SIV antigens of interest e.g., the gag, env, pol, sor, 3'orf antigen
  • a marker for the selection of recombinant vaccinia virus which may comprise:
  • a vaccinia promoter e.g., the BamF promoter of vaccinia virus linked to a gene encoding a selectable marker
  • vaccinia structural gene sequences which restore the function of the 29k polypeptide; e. DNA sequences homologous with a region of vaccinia nonessential for replication flanking the construct of elements a-d (e.g., the vaccinia TK or HindlllM sequence).
  • the intermediate DNA vectors containing the SIV gene(s) and the marker gene flanked by appropriate pox viral sequences undergo recombination with pox virus genomic DNA, which results in integration of the flanked gene into the viral genome.
  • Recombination occurs in a eukaryotic host cell.
  • Appropriate host cells for recombination are those which are 1) infectable by pox virus and 2) transfectable by the DNA vector. Examples of such cells are chick embryo fibroblast, CV-1 (monkey kidney cells), HuTK 143 cells (human cells), BSC40 (monkey kidney cells), RK-13 cells (rabbit kidney cells) and many others.
  • Viral infection is accomplished by standard techniques for infection of eukaryotic cells with pox virus.
  • the cells can be transfected with the intermediate vector by any of the conventional techniques of transfection. These include the technique of calcium phosphate precipitation, DEAE dextran, electroporation and protoplasm fusion. The preferred technique is the calcium phosphate precipitation technique. After infection and subsequent transfection, the cells are incubated under standard conditions and virus is allowed to replicate, during which time in vivo recombination occurs between the homologous pox virus sequences in the intermediate vector and the pox virus sequences in the genome.
  • Recombinant viral progeny are then identified by any of several techniques.
  • virus harboring the SIV gene(s) can be selected on the basis of inactivation of the viral gene into which foreign DNA was inserted.
  • the DNA vector is designed for insertion into the TK gene.
  • viruses containing integrated DNA will be TK and can be selected on this basis.
  • Preferred techniques for selection are based upon co-integration of a gene encoding a marker or indicator gene as described above.
  • One preferred indicator gene is the E. coli lacZ gene. Selection of recombinant viruses expressing B-galactosidase can be done by employing a chromogenic substrate for the enzyme.
  • a second preferred indicator gene is the vaccinia 29K gene. Selection of recombinant viruses that express the wild-type 29K gene-encoded function can be performed by growing the recombinant virus on RK-13 cells.
  • a variety of methods can be used to assay the expression of the polypeptide encoded by the inserted gene. These methods include black plaque assay (an in situ enzyme immunoassay performed on viral plaques), Western blot analyses, radioimmunoprecipitation (RIPA), immunofluorescence assays (IFA), and enzyme immunoassays (EIA).
  • black plaque assay an in situ enzyme immunoassay performed on viral plaques
  • Western blot analyses Western blot analyses
  • radioimmunoprecipitation (RIPA) immunofluorescence assays (IFA), and enzyme immunoassays (EIA).
  • the antibody used for these analyses is sera from macaque monkeys infected with SIV.
  • Recombinant viruses that contain genes encoding the envelope proteins of SIV can also be assayed for their ability to bind the CD4 receptor and induce the formation of syncytia in vitro.
  • Syncytia formation mimics, at least qualitatively, the processes involved in SIV entry into the host cell and can be used as a model to test the ability of an envelope polypeptide produced by vaccinia virus to function in this regard.
  • Recombinant vaccinia viruses that express SIV antigens can be initially evaluated in mice to determine the nature and magnitude of the humoral immune responses they elicit. Sera are assayed for the presence of antibodies specific for the expressed antigens and for the ability to neutralize SIV in vitro.
  • mice are inoculated with 10 pfu of recombinant virus by intraperitoneal injection. Serum samples obtained weekly for 4-6 weeks are assayed for anti-vaccinia titers using an enzymelinked immunoabsorbant assay (ELISA). The sera are then examined for specific response to the expressed antigens using the following assays:
  • ELISA For SIV recombinants, sera will be tested for SIV-specific humoral immune responses using a whole virus ELISA. For this assay, SIV purified from infected cells using a column chromotography procedure is used to coat the wells of microtiter plates, and the ELISA is performed using serial dilutions of the mouse sera.
  • Radioimmunoprecipitation (RIPA). RIPA can also be used to detect SIV-specific humoral immune responses. Radiolabelled whole SIV virus or SIV polypeptides expressed in heterologous mammalian expression systems can be used as the source of antigen for these studies.
  • a parameter believed to be critical for the ability of vaccine-induced antibodies to protect against virus challenge is the ability of the antibodies to neutralize virus infectivity.
  • Several methods are used to evaluate the neutralization capabilities of antibodies elicited by the vaccinia recombinants: a) Inhibition of syncytia formation. The ability of an antiserum to inhibit the formation of syncytia correlates with its ability to neutralize HIV virus infection. Fung et al. (1987) Biotechnology 5:940-946. Simian immunodeficiency virus is incubated with heat-activated murine sera, then inoculated onto c8166 (CD4+) cells. The presence of syncytia is scored at day 4 and at day 7.
  • vaccinia recombinants that express one or more SIV antigens and that elicit good levels of neutralizing antibodies in mice will be selected for detailed testing in macaque monkeys.
  • Vaccine Efficacy Studies in the SIV/Macaque Model System Testing of recombinant pox virus containing SIV DNA sequences encoding immunogenic SIV proteins includes measurement of total antibody response, of neutralizing antibody titers, of cell-mediated responses, and of the response of macaques vaccinated with the SIV/vaccinia recombinants to live SIV challenge.
  • the total antibody response in monkeys inoculated with recombinant vaccinia viruses expressing SIV antigens will be measured using a whole virus enzyme, linked immunosorbant assay (ELISA).
  • ELISA linked immunosorbant assay
  • SIV is purified from infected cells using a column chromatography procedure and ELISA plates are coated with whole lysed virus.
  • Antibodies in the same vaccinated monkey at different times and in different monkeys receiving the same vaccinia recombinant under different conditions can be compared in titer by their reaction in the whole virus ELISA.
  • SIV antigen SIV antigen
  • Both Western blot analysis and radioimmunoprecipitation (RIPA) can be used to identify the specific SIV antigen (s) recognized by antibodies from vaccinated macaques, both before and after challenge with live SIV.
  • Neutralization of virus infectivity The ability of vaccine-induced antibodies to neutralize viral infectivity is believed to be a parameter critical for protection against virus challenge.
  • a number of neutralization assays for SIV are currently used, including the 3 H-thymidine incorporation assay previously described.
  • Analysis of cell-mediated immunity SIVspecific proliferative responses, suppression of SIV outgrowth by CD8+ lymphocytes, and the nature and magnitude of cytotoxic T-lymphocyte responses can be measured.
  • the design of vaccination and challenge experiments is illustrated by the evaluation of vaccinia recombinants that express SIV env and gag polypeptides, in monovalent and divalent conformations.
  • the recombinants to be analyzed can be those that have expressed high levels of the inserted SIV gene products and that have elicited good anti-SIV immune responses in mice. 1. Macaque Monkeys
  • Juvenile rhesus macaques (Macaca mulatta) are chosen for inoculation with each vaccinia recombinant. Two age-matched rhesus macaques can serve as unvaccinated controls. The macaques can be pre-screened for good health, normal blood counts, and absence of anti-SIV antibodies. 2. Vaccination
  • the vaccination scheme can be as follows:
  • One macaque in each group can be inoculated with 10 8 PFU intradermally while the other can receive 10 9 PFU.
  • Blood samples can be taken at weeks 2 and 4 and vaccination with the same dose at a different site can be repeated at week 4.
  • Vaccination can be continued every four weeks until such time that anti-SIV antibody titers are no longer increased.
  • Humoral and cell-mediated immune responses measurements can be made as described in the previous section. These measurements are important for two reasons: 1) they provide preliminary evidence for the adequacy of the immune response; and 2) they serve to eventually identify the immune response parameters that correlate with protection. 4. Live SIV Challenge
  • vaccinated and control macaques can be challenged with a minimal dose of previously titered, cell-free, live, pathogenic SIV. It is important to use a minimal dose of cell-free virus because of the need to optimize the chance of successful protection in the initial experiments.
  • Various dilutions of the frozen stock SIV to be used for these experiments will have been inoculated previously into macaques; 1 ml of a 10 -4 dilution of this pathogenic stock virus (SIVmac251) can be administered intramuscularly for our initial challenges. (Clones derived from the SIVmac251 virus were used for the construction of the vaccinia recombinants).
  • E. coli stain MC1061 (Casadaban and Cohen, 1980, J. Mol. Biol. 138, 179) was used as the host for the growth of all plasmids.
  • the monkey kidney cell line BSC-40, the rabbit kidney cell line RK13 (Beale et al., (1963) Lancet, 2,640) and the thymidine kinase - deficient (TK-) human cell line Hu143TK- were used for vaccinia virus infections and transfections.
  • Vaccinia virus strain New York City Board of Health NYCBH
  • ATCC #VR-325 ATCC #VR-325
  • 29K- lacZ + strain vAbT33 See, U.S. Patent Application Serial No. 205,189, filed concurrently herewith
  • TK- recombinant plaques were selected and purified in the presence of 50 uM bromodeoxyuridine.
  • 29K recombinants were selected and purified on RK13 cells (see U.S. Patent Application Serial No. 205,189 , filed concurrently herewith).
  • DNA was extracted from vaccinia virus-infected cells as described (Esposito et al., J. Virol.
  • mice Female Balb/c mice were immunized I. P. with 10 - 10 8 pfu vaccinia virus recombinant. Sera were obtained three weeks post-immunization.
  • Anti-vaccinia antibody titers of sera from immunized mice were determined by enzyme-linked iinmunosorbant assay (ELISA), using vaccinia strain WR as antigen, performed essentially as described in U.S. Patent Application Serial No. 910,501, filed September 23, 1986.
  • ELISA enzyme-linked iinmunosorbant assay
  • Anti-SIV antibody titers of sera from immunized mice were determined by ELISA, using a whole-virus lysate from column-purified SIV. Daniel et al., (1988) Int. J. Cancer 41:601.
  • SIV neutralization titers of sera from immunized mice were determined by a 3 H-thymidine incorporation assay, which depends on the extreme sensitivity of the HTLV-transformed cell line MT-4 to the cytopathic effect of SIV and the ability of antisera with neutralizing activity to prevent these cytopathic effects.
  • SIV virus is incubated with the murine sera, then added to growing MT-4 cells. Infected cells are killed and do not incorporate 3 H- thymidine whereas cells protected from SIV infection do incorporate 3 H-thymidine. Daniel et al., (1988)
  • EXAMPLE 1 Recombination DNA Vectors Containing Vaccinia Promoters This Example illustrates construction of monovalent in vivo recombination vectors containing the vaccinia 40K or 30K promoter, with insertion at the vaccinia tk region ( Figures 2, 3).
  • the construction and structure of vectors pAbT4024, pAbT752, pAbT4500, pAG3, pAbT4007, have been described in U.S. Patent application Serial No. 910,501, filed September 23, 1986 incorporated herein by reference.
  • the vaccinia 40K promoter is located on the 8600bp vaccinia Hindlll H fragment (Rosel et al. (1986) J. Virology, 60:436-449).
  • a plasmid, pBR-Hind H, containing the Hindlll H fragment, was obtained from Bernard Moss (National Institutes of Health, Bethesda, MD).
  • pBR-Hind H was digested with FnuDII, and a 224bp fragment containing the 40K promoter was gel-purified.
  • the cloning vector DNA was an E. coli plasmid, derived from pBR322 (Bolivar et al.
  • pAbT2050 (1977) Gene, 2:95), denoted pAbT2050, the relevant features of which are a unique BamHI site, an E. coli replicon and an ampicillin-resistance gene (pAbT2050 also contains a lacZ gene but this is not required for the construction).
  • pAbT2050 was digested with BamHI, treated with Klenow, and was ligated to the 224 bp fragment to create pAbT4514, as shown in Figure 2A.
  • pAbT4514 was digested with BamHI, treated with Klenow, and a 230bp fragment containing the 40K promoter was gel-purified.
  • pEMBL18 (Dente et al. (1983) Nucl.
  • Acids Res., 11:1645) was digested with HincII and was ligated to the 230 bp fragment to create pAbT4514A, as shown in Figure 2B.
  • pAbT4514A was digested with Dral and BamHI, and a 161bp fragment containing the 40K promoter was gel-purified.
  • pEMBL18 was digested with HincIi and BamHI, and was ligated to the 161bp fragment to create pAbT4522, as shown in Figure 2C.
  • pAbT4024 was digested with Sphl and Kpnl, and a 420bp fragment containing the vaccinia 30K promoter (Perkus et al., (1985) Science, 229:881) was gelpurified.
  • pAbT752 was digested with Sphl and Kpnl, and a 7900bp fragment was gel-purified and ligated to the 420bp fragment to create pAbT4500, as shown in Figure 2D.
  • pAbT4500 is a plasmid vector for use in in vivo recombination experiments in vaccinia.
  • pAbT4500 contains the vaccinia TK region for directing recombination, a lacZ gene under the control of the vaccinia BamF promoter for identification of vaccinia recombinants, the 30K promoter followed by a multiple cloning site for the insertion and expression of foreign antigens, and a bacterial replicon and ampicillin-resistance gene for growth and selection in E. coli.
  • pAbT4522 was digested with Sphl and Kpnl, and a 170bp fragment containing the 40K promoter was gel-purified.
  • pAbT4500 was digested with Sphl and Kpnl, and a 7900bp fragment was gel-purified and ligated to the 170bp fragment to create pAbT4525, as shown in Figure 2E.
  • pAbT4525 is also a plasmid vector for use in in vivo recombination experiments in vaccinia.
  • pAbT4525 is identical to pAbT4500, except that the 4OK promoter is substituted for the 30K promoter.
  • pAbT4525 was slightly modified as follows: pAG3 was digested with Ndel, treated with Klenow, and ligated to Notl linkers (New England BioLabs; dGCGGCCGC) to create pAbT4524A, as shown in Figure 3A. pAbT4524A was digested with Notl, treated with Klenow, and ligated to Dral linkers (Biology Dept., Brandeis University; dGCTTTAAAGC) to create pAbT4524B, as shown in Figure 3B. Notl sites were expected to be regenerated both 5' and 3' to the Dral site; due to exonuclease digestion, the Notl site 3' to the Dral site was not regenerated.
  • pAbT4007 was digested with Dral, and a 5300bp fragment containing the vaccinia 7.5K promoter, the lacZ gene under the control of the BamF promoter, and most of the tk flanking regions, was gelpurified.
  • pAbT4524B was digested with Dral and was ligated to the 5300bp fragment to create pAbT4532B, as shown in Figure 3C.
  • pAbT4525 was digested with Sphl and Kpnl, and the 170bp fragment containing the vaccinia 40K promoter was gel-purified.
  • pAbT4532B was digested with Sphl and Kpnl, and a 7300bp fragment was gel-purified and ligated to the 170bp fragment to create pAbT4537, as shown in Figure 3D.
  • pAbT4537 is a plasmid vector for use in IVR experiments in vaccinia.
  • pAbT4537 is identical to pAbT4525, except that the tk flanking regions were shortened and modified to contain additional, convenient restriction sites.
  • EXAMPLE 2 Recombination Vector Containing the 30K Vaccinia Promoter
  • This Example illustrates the construction of a monvalent in vivo recombination vector containing the vaccinia 30K promoter, with insertion at the vaccinia Hindlll M region ( Figure 4).
  • the construction and structure of vectors pAbT3101 and pAbT3100 have been described in co-pending application Serial No. 910,501, incorporated herein by reference.
  • pAbT3101 was digested with EcoRI and SalI, and a 420bp fragment containing the vaccinia 30K promoter was gel-purified.
  • pAbT3100 containing the vaccinia Hindlll M fragment, was digested with Sphl, treated with T4 DNA polymerase, ligated to EcoRI linkers (New England BioLabs), then digested with SalI, and a 3600bp fragment was gel-purified. The 3600bp and 420bp fragments were ligated to create pAbT3105, as shown in Figure 4A.
  • pAbT3105 was digested with EcoRI and ligated to BamHI linkers (New England BioLabs) to create pAbT3106, as shown in Figure 4B.
  • pAbT3106 was digested with BamHI and ligated to SacI linkers (New England BioLabs) to create pAbT3106S, as shown in Figure 4C.
  • pAbT4500 was digested with Sphl and Kpnl, and a 420bp fragment containing the vaccinia 30K promoter was gel-purified.
  • pAbT4532B was digested with Sphl and Kpnl, and a 7300bp fragment was gel-purified and ligated to the 420bp fragment to create pAbT4536, as shown in Figure 4D.
  • pAbT4536 was digested with SalI and SacI, and a 420bp fragment containing the 30K promoter was gel-purified.
  • pAbT3106S was digested with SalI and SacI, and a 3500bp fragment was gel-purified and ligated to the 420bp fragment to create pAbT4554, as shown in Figure 4E.
  • pAbT4554 is a plasmid vector for use in IVR experiments in vaccinia.
  • pAbT4554 contains the vaccinia Hindlll M region for directing recombination, including a portion of the 29K host-range gene for selection of recombinants, the 30K promoter followed by a multiple cloning site for the insertion and expression of foreign antigens, and a bacterial replicon and ampicillin-resistance gene for growth and selection in E. coli.
  • EXAMPLE 3 Construction of a divalent in vivo recombination vector containing the vaccinia 30K and 40K promoters pAbT4537 was digested with EcoRI and BamHI, and a 200bp fragment containing the vaccinia 40K promoter was gel-purified. pAbT4554 was partially digested with EcoRI and completely digested with BamHI and was ligated to the 200bp fragment to create pAbT4555, as shown in Figure 5A. pAbT4555 is a divalent vector for use in IVR experiments in vaccinia.
  • pAbT4555 contains the vaccinia Hindlll M region for directing recombination, the vaccinia 29K host range gene for selection of recombinants, two vaccinia promoters, 30K and 40K, each followed by multiple cloning sites for the insertion and expression of foreign antigens, and a bacterial replicon and ampicillin-resistance gene for growth and selection of E coli.
  • EXAMPLE 4 Trivalent in vivo recombination vector containing the vaccinia 30K, 40K and 7.5K promoters pAbT4532B was digested with Smal and Xbal, treated with Klenow and self-ligated to create pAbT4532X, as shown in Figure 5B.
  • pAbT4532X was digested with HincII, ligated to Smal linkers, digested with Smal and SacI, and a 270bp fragment containing the vaccinia 7.5K promoter was gelpurified.
  • pAbT4555 was digested with Smal and SacI, and was ligated to the 270bp fragment to create pAbT4556, as shown in Figure 5C.
  • pAbT4556 is a trivalent vector for use in in vivo recombination experiments in vaccinia.
  • pAbT4556 is identical to pAbT4555, except pAbT4556 contains three vaccinia promoters, 30K, 40K and
  • EXAMPLE 5 Construction of a divalent in vivo recombinant vector containing the vaccinia 7.5K and 40K promoters pAbT4027 (U.S. patent application, Serial No. 910,501) was digested with Dral and a 5530 bp fragment is gel-purified.
  • pAbT4524B was partially digested with Dral.
  • a 2260 bp fragment was gel- purified and ligated to the 5530 bp fragment to create pAbT4533, as shown in Figure 6A.
  • pAbT4556 was digested with Sphl and Smal, and a 200bp fragment containing the vaccinia 40K promoter was gel-purified.
  • pAbT4533 was digested with Xbal, treated with Klenow, and ligated to Xhol linkers (New England BioLabs; dCCTCGAGG) to create pAbT4533X, as shown in Figure 6B.
  • pAbT4533X was digested with Sphl and Smal, and a 7400bp fragment was gel-purified and ligated to the 200bp fragment to create pAbT4586, as shown in Figure 6C.
  • pAbT4586 is a divalent vector for use in IVR experiments in vaccinia.
  • pAbT4586 contains the vaccinia TK gene region for directing recombination, the BamF-lacZ gene for identification of vaccinia recombinants, the 7.5K and 40K promoters, each followed by a multiple cloning site for the insertion and expression of foreign antigens, and a bacterial replicon and ampicillin-resistance gene for growth and selection in E.coli.
  • EXAMPLE 6 Construction of a monovalent in vivo recombination vector containing the SIV MAC-251 gene encoding envelope protein gp160 under the control of the vaccinia 40K promoter
  • Bacteriophage lambda EMBL4 containing fulllength proviral SIV macaque strain 251 (SIV MAC-251 ) DNA was obtained from Ronald C. Desrosiers (New England Regional Primate Research Center (NERPRC), Southborough, MA). This DNA, denoted lambda SIV 251 , was digested with SacI and a 3500bp fragment containing the gp160-encoding region (env) was gelpurified.
  • pEMBL18 (Dente et al., (1983) Nucl. Acids Res. 11:1645) was digested with SacI and was ligated to the 3500bp fragment to create pAbT4566, as shown in Figure 7A.
  • pAbT4566 was partially digested with PvuII, and a 6500bp fragment containing all but the 5' 21bp of env and the 5' flanking region of env was gelpurified and ligated to DT210 and DT211 to create pAbT4096, as shown in Figure 7B.
  • pAbT4096 The env gene in pAbT4096 now has an Xbal site and an Ncol site 7 and lbp, respectively, 5' to the env initiation codonATG.
  • pAbT4096 was digested with Xbal and SacI, and a 2600bp fragment was gel-purified.
  • pAbT4537 was partially digested with SacI and was completely digested with Xbal. The resulting 7300bp fragment was gel-purified and ligated to the 2600bp fragment to create pAbT4572, as shown in Figure 7C.
  • pAbT4572 is a vector for the insertion and expression of SIV MAC-251 env in vaccinia.
  • pAbT4572 contains the env gene under the control of the vaccinia 40K promoter, the DNA regions flanking the vaccinia TK gene for directing recombination in vaccinia, the lacZ gene under the control of the vaccinia BamF promoter for selection of vaccinia recombinants and a bacterial replicon and ampicillinresistance gene for growth and selection in E.coli ( Figure 7C).
  • Plasmid pHS251 containing the central portion of the SIV MAC-251 Proviral genome, was obtained from Ronald C. Desrosiers (NERPRC). This DNA was digested with Kpnl, treated with T4 DNA polymerase, then was digested with Scal, and a 2200bp fragment containing the gag gene was purified. This fragment also contains 900bp of the gene encoding the pol polyprotein, including the entire coding sequence of the protease required for the processing of the gag polyprotein; this fragment is therefore denoted as containing the gag-prot gene.
  • NERPRC Ronald C. Desrosiers
  • pAbT4537 was digested with Smal and was ligated to the 2200bp fragment to create pAbT4575 as shown in Figure 8A.
  • pAbT4575 is a vector for the insertion and expression of SIV MAC-251 gag and protease in vaccinia.
  • pAbT4575 is identical to pAbT4572 described in Example 6, except that pAbT4575 contains the gag-prot gene under the control of the vaccinia 40K promoter ( Figure 8A).
  • SIV MAC_-251 env gene under the control of the vaccinia 30K promoter and the SIV MAC- 251 gag-prot gene under the control of the vaccinia 40K promoter pAbT4572 was digested with Xbal, treated with Klenow, then digested with SacI, and a 2600bp fragment containing the env gene was gel-purified.
  • pAbT4554 was digested with Smal and SacI, and was ligated to the 2600bp fragment to create pAbT4574, as shown in Figure 8B.
  • pAbT4574 was partially digested with EcoRI and was completely digested with SalI, and a 3000bp fragment containing the 30K promoter and env gene was gel-purified.
  • pAbT4575 was digested with EcoRI and SacI, and a 2400bp fragment containing the 40K promoter and the gag-prot gene was gel-purified.
  • pAbT4555 was digested with SalI and SacI, and a 3500bp fragment was gel-purified and ligated to the 3000bp and 2400bp fragments to create pAbT4577, as shown in Figure 8C.
  • pAbT4577 is a vector for the insertion and expression of SIV MAC-251 env and gag-prot in vaccinia.
  • pAbT4577 contains the env gene under the control of the vaccinia 30K promoter and the gagprot gene under the control of the vaccinia 40K promoter, flanked by vaccinia DNA for directing recombination into the vaccinia Hindlll M region.
  • the vector DNA includes the 29K host-range gene for selection of vaccinia recombinants and a bacterial replicon and ampicillin-resistance gene for growth and selection in E.coli ( Figure 8C).
  • EXAMPLE 9 Construction of a monovalent in vivo recombination vector containing the S IV MAC-251 3'ORF gene under the control of the vaccinia 30K promoter
  • Plasmid p251PR3 'ORF-I containing a portion of of the SIV MAC-251 proviral genome including the gene encoding 3'ORF, was obtained from Ronald C. Desrosiers (NERPRC).
  • p251PR3'ORF-1 was digested with Pstl and Stul, and an 800bp fragment containing the 3'ORF gene was gel-purified.
  • pAbT4555 was digested with Pstl and Smal, and a 3900bp fragment was gelpurified and ligated to the 800bp fragment to create pAbT4576, as shown in Figure 9A.
  • pAbT4576 is a vector for the insertion and expression of SIV MAC-251 3'ORF in vaccinia.
  • pAbT4576 contains the 3'ORF gene under the control of the vaccinia 30K promoter and contains the same vaccinia recombination and selection elements and bacterial components described for pAbT4577 in Example 8 ( Figure 9A).
  • EXAMPLE 10 Construction of a monovalent in vivo recombination vector containing the SIV MAC-251 sor gene under the control of the vaccinia 30K promoter Lambda SIV 251 DNA was digested with Hindlll, and a 4300bp fragment containing a portion of the SIV MAC-251 proviral genome including the pol and sor genes was gel-purified.
  • pEMBL18 was digested with Hindlll and was ligated to the 4300bp fragment to create pAbT4579, as shown in Figure 10A.
  • pAbT4579 was digested with HincIi and Hphl, treated with T4 DNA polymerase, and a 1000bp fragment was gel-purified.
  • pAbT4554 was digested with Smal and was ligated to the 1000bp fragment to create pAbT4581, as shown in Figure 10B.
  • pAbT4581 is a vector for the insertion and expression of the SIV MAC-251 sor gene in vaccinia.
  • pAbT4581 is identical to pAbT4576 described in
  • SIV MAC-251 pol gene under the control of the vaccinia 40K promoter pAbT4579 was digested with Hindlll and Pstl, and a 1200bp fragment, containing the 5' end of the pol gene, was gel-purified.
  • Bacteriophage m13mp18 DNA (New England BioLabs) was digested with Hindlll and Pstl, and was ligated to the 1200bp fragment to create pAbT4579A, as shown in Figure 11A.
  • the 5' end of the pol gene was modified to insert a convenient restriction site and a translation initiation ATG codon, by oligonucleotidedirected mutagenesis, as described in Materials and Methods.
  • oligonucleotide Bossarcoma
  • Figure 11B Using the oligonucleotide (Biology Dept., Brandeis University) shown in Figure 11B, a BamHI site and ATG codon were inserted at the beginning of the pol-encoding region, creating pAbT4579B, as shown in Figure 11B.
  • pAbT4579B was digested with BamHI and Pstl, and a 900bp fragment containing the 5' end of pol was gel-purified.
  • Bacteriophage ml3mpl9 DNA (New England BioLabs) was digested with BamHI and Pstl, and was ligated to the 900bp fragment, to create pAbT4579C, as shown in Figure lie
  • pAbT4579 was digested with Pstl, and a 2400bp fragment containing the 3' end of pol was gelpurified.
  • pAbT4579C was digested with Pstl, and was ligated to the 2400bp fragment, to create pAbT4582, as shown in Figure 11D.
  • pAbT4582 was digested with BamHI and Hindlll, and the 3300bp fragment containing the modified pol gene was gel-purified.
  • pEMBL18 was digested with BamHI and Hindlll, and was ligated to the 3300bp fragment to create pAbT4582B, as shown in Figure 11E.
  • pAbT4582B was digested with Hindlll, treated with Klenow, ligated to SacI linkers (New England BioLabs), digested with SacI, and a 3300bp fragment was gel-purified.
  • pAbT4537 was partially digested with SacI and was ligated to the 3300bp fragment to create pAbT4583, as shown in Figure 11F.
  • pAbT4583 is a vector for the insertion and expression of SIV MAC-251 pol in vaccinia.
  • pAbT4583 is identical to pAbT4572 described in Example 6, except that pAbT4583 contains the pol gene under the control of the vaccinia 40K promoter ( Figure 11F).
  • EXAMPLE 12 Construction of a divalent in vivo recombination vector
  • This vector contains the SIV MAC-251 env gene under the control of the vaccinia 40K promoter and the SIV MAC-251 gag-prot gene under the control of the vaccinia 7.5K promoter, with insertion at the vaccinia Hindlll M region.
  • pAbT4575 was digested with BamHI and SacI, and a 1300bp fragment containing the 3' portion of the gag-prot gene was gel-purified.
  • pHS251 was digested with Kpnl and BamHI, and a 900bp fragment containing the 5' portion of the gag gene was gel-purified.
  • pAbT4554 was digested with Kpnl and Sacl, and was ligated to the 1300bp and 900bp fragments to create pAbT4578, as shown in Figure 12A.
  • pAbT4574 was digested with BamHI, and a 2600bp fragment containing the env gene was gel-purified.
  • pAbT4556 was digested with BamHI and was ligated to the 2600bp fragment to create pAbT4556A, as shown in Figure 12B.
  • pAbT4578 was digested with Kpnl and SacI, and a 2600bp fragment containing the gag-prot gene was gel-purified.
  • pAbT4556A was digested with Kpnl and Sacl, and was ligated to the 2600bp fragment to create pAbT4585, as shown in Figure 12C.
  • pAbT4585 is a vector for the insertion and expression of the SIV MAC-251 env and gag-prot genes in vaccinia.
  • pAbT4585 is identical to pAbT4577, except that pAbT4585 contains the env gene under the control of the 40K promoter and the gag-prot gene under the control of the 40K promoter and the gag-prot gene under the control of the 7.5K promoter ( Figure 12C).
  • EXAMPLE 13 Construction of a divalent in vivo recombination vector
  • This vector contains the SIV MAC-251 env gene under the control of the vaccinia 40K promoter and the SIV MAC-251 gag-prot gene under the control of the vaccinia 7.5K promoter, with insertion at the vaccinia TK region.
  • pAbT4532B was partially digested with EcoRI and completely digested with Hindlll, and a 3100bp fragment containg the lacZ gene was gel-purified.
  • pAbT4585 was digested with EcoRI and SacI, and a 5600bp fragment containing the vaccinia 40K promoter, the SIV MAC-251 env gene, the vaccinia 7.5K promoter and the SIV MAC-251 gag-Prot gene was gel-purified.
  • pAbT4586 was digested with SacI and Hindlll and a 4200bp fragment was gel-purified and ligated to the 3100bp and 5600bp fragments to create pAbT4589, as shown in Figure 13A.
  • pAbT4589 is a vector for the insertion and expression of SIV MAC-251 env and gag-prot in vaccinia.
  • pAbT4589 contains the SIV env gene under the control of the vaccinia 40K promoter, the SIV gag-prot gene under the control of the vaccinia 7.5K promoter, the DNA regions flanking the vaccinia TK gene for directing recombination in vaccina, the lacZ gene under the control of the vaccinia BamF promoter for selection of vaccinia recombinants and a bacterial replicon and ampicillin-resistance gene for growth and selection in E.coli ( Figure 13A).
  • EXAMPLE 14 Construction of recombinant vaccinia viruses containing SIV MAC 251 genes under the control of vaccinia promoters.
  • In vivo recombination is a method whereby recombinant vaccinia viruses are created (Nakano et al., (1982) Proc. Natl. Acad. Sci. USA 79:1593; Paoletti and Panicali, U.S. Patent No. 4,603,112). These recombinant viruses are formed by transfecting DNA containing a gene of interest into cells which have been infected by vaccinia virus. A small percent of the progeny virus will contain the gene of interest integrated into a specific site on the vaccinia genome. These recombinant viruses can express genes of foreign origin (Panicali and Paoletti. (1982) Proc. Natl. Acad. Sci. USA 79:
  • SIV MAC-251 genes were inserted into the vaccinia virus genome at either the Hindlll J or Hindlll M region, as listed in Table 2.
  • Vaccinia Vaccinia Promoter Vaccinia Vaccinia Promoter Recombinant SIV MAC-251 Gene(s) Recombinant SIV MAC _ 251 Gene(s) vAbT193 40K env vAbT198 30Kenv+40Kgag-prot vAbT197 40K gag-prot vAbT199 30K 3' orf vAbT222 40K pol vAbT206 30K sor vAbT239 40K env+7.5Kgag-prot vAbT223 40Kenv+7.5gag-prot
  • the TK gene is located in the Hindlll J region.
  • IVR vectors pAbT4572, 4575, 4583 or 4589 were transfected into Hu143TK- cells which had been infected with the NYCBH (TK + ) strain of vaccinia virus (see Materials and Methods).
  • the selection system for recombinant virus was bromodeoxyuridine (BUdR), which is lethal for TK + virus but allows recombinant, TK- virus to grow.
  • recombinant virus contains the lacZ gene which will metabolize Bluo-Gal and turn recombinant plaques blue.
  • vAbT193 contains env; vAbT197, gag-prot; vAbT222, pol; vAbT239, env + gag-prot.
  • the 29K host-range gene is located in the Hindlll M region (Gillard et al., (1986) Proc. Natl. Acad. Sci. USA 83: 5573) and has been developed as a selection system.
  • Recombinant vaccinia virus vAbT33 contains the lacZ gene in place of the 29K gene. Therefore vAbT33 cannot grow on RK13 cells which require the 29K gene product, and, on permissive cells, is blue in the presence of BluoGal. See U.S. Patent Application Serial No. 205,189, filed concurrently herewith.
  • IVR vectors pAbT4577, 4576, 4581 or 4585 were transfected into BSC-40 cells which had been infected with vaccinia virus vAbT33 (See Materials and Methods). Recombinant viruses were selected as white plaques in the presence of BluoGal on RK13 cells. Plaques were picked and purified, and were shown, by Southern analysis, to contain the appropriate SIV MAC-251 gene(s) : vAbT198 contains env + gag-prot; vAbT199, 3' orf; vAbT206, sor; vAbT223, env + gag-prot.
  • EXAMPLE 15 Black plaque assay for expression of S IV MAC-251 antigens in recombinant vaccinia.
  • the black plaque assay is an in situ enzyme-based immunoassay which can detect protein expressed by vacciniainfected cells.
  • the black plaque assay was performed on vaccinia recombinants vAbT193, 197, 198, 199, 206, 222 and 223, using SIV-infected macaque serum 127-87 obtained from Ronald C. Desrosiers (NERPRC).
  • Each recombinant contains SIV MAC-251 Proteins of the predicted correct sizes: the env-containing recombinants contain the precursor gp160 and the processed gp120/gp32; the gag-prot-containing recombinants contain predominantly the precursor p55 but also the 40K intermediate and the processed p24 and p15; the pol-containing recombinants contain the p64, p53 and p10 processed products. Thus each of these vaccinia recombinants is expressing the appropriate SIV MAC-251 antigen and. is also undergoing correct processing of these antigens.
  • EXAMPLE 17 Enzyme-linked immunosorbant assay (ELISA) with immune sera of mice immunized with recombinant vaccinia virus expressing SIV MAC-251 antigens
  • mice were immunized with vaccinia recombinants vAbT193, 197, 198 or 223, as described in Materials and Methods. Sera were obtained three weeks post-immunization and tested against vaccinia or against SIV by ELISA, as described in Materials and Methods. The results are shown in Table 3. Most mice exhibited a good anti-vaccinia immune response against the vaccinia recombinants, and a good anti-SIV response was observed with vaccinia recombinants vAbT198 and 223.
  • mice sera described in Example 17 were tested in an SIV neutralization assay, as described in Materials and Methods. Results are shown in Table 4. Antisera from all the vAbT198-immunized mice and one vAbT223- immunized mouse efficiently neutralized SIV; antisera from the vAbT197-immunized mice exhibited lower neutralization activity; the other antisera did not neutralize SIV.
  • vAbT223 C1 1:1600 1 : 1280 1:20 40K env C2 1:50 ⁇ 1 : 20 1:20 7.5K gag C3 1:6400 1 : 2560 1:1280 C4 ⁇ 1:50 1 : 20 ⁇ 1:20
  • the recombinant plasmid pAbT 4585 has been placed on deposit at the American Type Culture Collection in Rockville, Maryland, U.S.A.
  • the plasmid has been assigned the accession number 67729.

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Abstract

Cette invention concerne des virus de la variole recombinants capables d'exprimer des antigènes viraux d'immunodéficience simiens et de mettre en lumière une réaction immune protectrice contre le VIS (virus d'immunodéficience simien). L'invention concerne en outre un système de modèle de primate non humain utilisant des macaques servant à évaluer l'efficacité avec laquelle le virus de la variole recombinant exprimant des antigènes de VIS peut protéger les macaques contre l'acquisition et le développement du SIDA simien. L'importance du système de modèle de primate non humain et des procédés permettant de générer des virus de la variole recombinants contenant des gènes du VIS et leurs homologues du VIH, réside dans la mise au point de virus de la vérole recombinants exprimant une combinaison de gènes ou d'épitopes du VIH-1 permettant une protection totale à long terme contre l'infection du VIH-1 chez l'homme. L'utilisation du système de modèle animal de cette invention pour l'évaluation d'un vaccin permet de préparer des vaccins destinés à être utilisés dans des essais effectués sur l'homme.
PCT/US1989/002485 1988-06-10 1989-06-12 Procede d'evaluation de vaccins recombinants contre le virus de l'immunodeficience WO1989012095A1 (fr)

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WO1992010578A1 (fr) * 1990-12-13 1992-06-25 Bioption Ab Systemes d'expression de l'adn bases sur les alphavirus
US5571712A (en) * 1989-10-13 1996-11-05 Connaught Laboratories Limited Non-infectious, replication defective, immunogenic HIV retrovirus-like particles produced from a recombinant HIV genome devoid of long terminal repeats
US5674984A (en) * 1990-04-03 1997-10-07 Genentech, Inc. Method for isolation of unclipped HIV envelope protein
US5864027A (en) * 1993-06-07 1999-01-26 Genentech, Inc. HIV envelope polypeptides
EP1288304A2 (fr) * 1989-06-01 2003-03-05 Applied Biotechnology, Inc. Particules virales auto-assemblées, defectives, non auto-propagatrices
US6585979B1 (en) 1996-07-08 2003-07-01 Genentech, Inc. HIV envelope polypeptides and immunogenic composition
EP1372710A2 (fr) * 2001-03-08 2004-01-02 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Mva exprimant des genes enveloppe, gag et pol du vih
US6770283B1 (en) 1990-12-13 2004-08-03 Bioption Ab DNA expression systems based on alphaviruses
US7041293B1 (en) 1990-04-03 2006-05-09 Genentech, Inc. HIV env antibodies
US7795017B2 (en) 2000-03-02 2010-09-14 Emory University DNA expression vectors and methods of use
US8623379B2 (en) 2000-03-02 2014-01-07 Emory University Compositions and methods for generating an immune response

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JP3504659B2 (ja) * 1991-06-14 2004-03-08 ヴァイロジェネティクス コーポレイション 免疫不全ウイルス組換えポックスウイルスワクチン

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EP0245136A1 (fr) * 1986-04-08 1987-11-11 Transgene S.A. Vecteur viral, codant pour une glycoprotéine du virus responsable du SIDA, vaccin et anticorps

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EP0243029A1 (fr) * 1986-04-08 1987-10-28 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Virus vaccinia recombinant, exprimant un gène d'un rétrovirus humain
EP0245136A1 (fr) * 1986-04-08 1987-11-11 Transgene S.A. Vecteur viral, codant pour une glycoprotéine du virus responsable du SIDA, vaccin et anticorps

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Science, Volume 244; 12 May 1989, M.L. BOSCH et al.: "Indentification of the Fusion Peptide of Primate Immunodeficiency Viruses", pages 694-697 *

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EP1288304A3 (fr) * 1989-06-01 2006-04-05 Applied Biotechnology, Inc. Particules virales auto-assemblées, defectives, non auto-propagatrices
EP1288304A2 (fr) * 1989-06-01 2003-03-05 Applied Biotechnology, Inc. Particules virales auto-assemblées, defectives, non auto-propagatrices
US5985641A (en) * 1989-10-13 1999-11-16 Connaught Laboratories Limited Non-infectious, replication-impaired, immunogenic human immunodeficiency virus type 1 retrovirus-like particles with multiple genetic deficiencies
US5571712A (en) * 1989-10-13 1996-11-05 Connaught Laboratories Limited Non-infectious, replication defective, immunogenic HIV retrovirus-like particles produced from a recombinant HIV genome devoid of long terminal repeats
US6291227B1 (en) 1989-10-13 2001-09-18 Aventis Pasteur Limited Nucleic acids containing modified human immunodeficiency virus genomes devoid of long terminal repeats encoding non-infectious, replication-deficient, immunogenic retrovirus-like particles
US7041293B1 (en) 1990-04-03 2006-05-09 Genentech, Inc. HIV env antibodies
US5849533A (en) * 1990-04-03 1998-12-15 Genetech, Inc. Method for making unclipped HIV envelope protein
US5674984A (en) * 1990-04-03 1997-10-07 Genentech, Inc. Method for isolation of unclipped HIV envelope protein
WO1992010578A1 (fr) * 1990-12-13 1992-06-25 Bioption Ab Systemes d'expression de l'adn bases sur les alphavirus
US6770283B1 (en) 1990-12-13 2004-08-03 Bioption Ab DNA expression systems based on alphaviruses
US6190666B1 (en) 1990-12-13 2001-02-20 Bioption DNA expression systems based on alphaviruses
US5739026A (en) * 1990-12-13 1998-04-14 Bioption Ab DNA expression systems based on alphaviruses
US5864027A (en) * 1993-06-07 1999-01-26 Genentech, Inc. HIV envelope polypeptides
US6806055B2 (en) 1993-06-07 2004-10-19 Genentech, Inc. HIV envelopolype peptides
US6042836A (en) * 1993-06-07 2000-03-28 Genentech, Inc. HIV envelope polypeptides
US6331404B1 (en) 1993-06-07 2001-12-18 Genentech, Inc. HIV envelope polypeptides
US6585979B1 (en) 1996-07-08 2003-07-01 Genentech, Inc. HIV envelope polypeptides and immunogenic composition
US7071322B2 (en) 1996-07-08 2006-07-04 Genentech, Inc. HIV envelope polynucleotides and immunogenic composition
US7795017B2 (en) 2000-03-02 2010-09-14 Emory University DNA expression vectors and methods of use
US8623379B2 (en) 2000-03-02 2014-01-07 Emory University Compositions and methods for generating an immune response
EP1372710A2 (fr) * 2001-03-08 2004-01-02 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Mva exprimant des genes enveloppe, gag et pol du vih
EP1372710A4 (fr) * 2001-03-08 2005-04-27 Us Gov Health & Human Serv Mva exprimant des genes enveloppe, gag et pol du vih
US7867982B2 (en) 2001-03-08 2011-01-11 Emory University MVA expressing modified HIV envelope, gag, and pol genes
US8916172B2 (en) 2001-03-08 2014-12-23 Emory University MVA expressing modified HIV envelope, gag, and pol genes

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